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In the phylum Placozoa, only two species have been described, Trichoplax adhaerens Schulze (1883) and Treptoplax reptans Monticelli (1893). Whereas the former has been reported from tropical and subtropical localities around the world, the latter has not been observed since Monticelli's original description, and its validity is generally doubted. Thus, the high level of genetic diversity in our samples from a relatively small number of localities is quite surprising. Four separate molecular markers (nuclear 18S, ITS1-5.8S-ITS2, and 28S, and mitochondrial 16S)indicate that Placozoa comprises at least four distinct, deeply divided clades, all of which fit the gross morphological description of T. adhaerens. Whereas molecular differences within the four groups are modest, differences between the four clades appear large (for example, partial 16S lengths range from 544 to 689 bp). The deep divisions between the clades are comparable to those seen between different families or orders of cnidarians, suggesting a similarly long history. So far, no straightforward relationship has emerged between placozoan clades and biogeography or habitat. For instance, multiple groups are present on both the Pacific and Caribbean sides of Panama, in two cases at the same site, but a single group dominates our samples from both regions. Continued sampling from diverse localities and habitats from around the world promises to provide a more accurate picture of biodiversity in this supposedly monotypic phylum, and scrutiny of the ultrastructure of placozoans in the four clades may reveal diagnostic differences in morphology. Indeed, the discovery of a large complex of nested placozoan taxa now seems inevitable.

The placozoan Trichoplax adhaerens displays the s mplest morphology of any free-living animal, can be easily maintained in the laboratory, and possesses the smallest genome of any animal yet measured (50 Mb). A consortium of marine invertebrate biologists, geneticists and genomic scientists has been assembled to begin the task of genome sequencing, annotation and gene analysis of the Trichoplax genome. We will report on biological, genomic and informatic resources now under development for this system. A geographically diverse collection of laboratory strains has been established. The Trichoplax Genome Project has completed a confirmation of the genome size, gene density estimates, AT:GC content. The composition of several large contiguous genomic regions and the mitochondrial genome sequence and content. An arrayed fosmid library and small-insert genomic library has been finished and a full-length cDNA library is currently under construction for EST and microarray investigations. Collectively these efforts are expected to collectively generate the suite of resources expected in a modern experimental model.

A more than a century old debate has been focusing on the origin of the Metazoa and the hypothetical urmetazoan bauplan. A basal position for the morphologically most simply organized metazoan animal, the placozoan Trichoplax adhaerens, was widely accepted for almost a century. The limited number of only four somatic cell types, the smallest metazoan genome, and the lack of any foot or head structures, of any anterior-posterior organization, any kind of organs, and of both a basal lamina and an extracellular matrix (ECM) placed Trichoplax in a basal and isolated position relative to all other metazoan phyla. Butschlis placula hypothesis of the urmetazoon was based on this view and provided a hypothetical scenario for both, the evolution of diploblast and triploblast bauplans. We have been analysing (i) the sum of morphological data, (ii) the molecular sequence data from two mitochondrial genes, COII and COIII, (iii) the molecular sequence data from two regulatory nuclear genes, Cnox-2 and the PaxB gene, (iv) the secondary structure of the 16S rRNA molecule, and (v) the putative cluster organization of Hox genes. The evidence strongly supports the view that Placozoa are basal relative to all other recent diploblast phyla. Furthermore, the comparison of Hox/ ParaHox gene expression patterns provide an intriguing hypopthetical scenario for Butschlis original placula hypothesis and thus the hypothetical urmetazoan bauplan.

Cnidaria has been the subject of several analyses of molecular data that attempt to resolve evolutionary relationships among and within its component groups. These studies have renewed interest in cnidarian character evolution and may be partly responsible for spurring the numerous investigations of cnidarian relationships based on morphological and life history features. This bounty of analyses aimed at achieving the single best phylogenetic hypothesis has produced an unprecedented number of alternative ideas about the evolutionary history of Cnidaria. Great uncertainty would appear to exist when only a short time ago we enjoyed unprecedented optimism concerning the tractability of phylogenetic questions. However, independently proposed phylogenetic trees based on diverse data, when looked at as a set of hypotheses, show strong congruence of results. We further clarify the emerging view of cnidarian phylogeny by using nearly complete 28S rDNA sequences (roughly 3,300 bp). Diverse data now indicate that Hydrozoa and Cubozoa are monophyletic, while Scyphozoa is not. The sessile members of Stauromedusae are likely to be the sister group of all other medusozoans. Thus, the ancestral medusozoan was probably solitary, biradial in its early ontogeny, with four intramesogleal muscles of ectodermal origin associated with peristomial pits surrounding the mouth. From this state, hydrozoan and cubozoan polyps are probably simplified. The ancestral hydrozoan probably produced medusae with a velum via lateral budding from polyps and development involving an entocodon. Gastric filaments, hollow sensory structures derived ontogenetically from tentacles, a circular coronal muscle, and septa of the medusa probably were lost in the ancestry of Hydrozoa, perhaps indicating (though not confirming) independent origins of the medusa.

There are considerable phylogenetic incongruencies between morphological and phylogenomic data for the deep evolution of animals. This has contributed to a heated debate over the earliest-branching lineage of the animal kingdom: the sister to all other Metazoa (SOM). Here, we use published phylogenomic data sets ($\sim $45,000-400,000 characters in size with $\sim $15-100 taxa) that focus on early metazoan phylogeny to evaluate the impact of incorporating morphological data sets ($\sim $15-275 characters). We additionally use small exemplar data sets to quantify how increased taxon sampling can help stabilize phylogenetic inferences. We apply a plethora of common methods, that is, likelihood models and their "equivalent" under parsimony: character weighting schemes. Our results are at odds with the typical view of phylogenomics, that is, that genomic-scale data sets will swamp out inferences from morphological data. Instead, weighting morphological data 2-10$\times $ in both likelihood and parsimony can in some cases "flip" which phylum is inferred to be the SOM. This typically results in the molecular hypothesis of Ctenophora as the SOM flipping to Porifera (or occasionally Placozoa). However, greater taxon sampling improves phylogenetic stability, with some of the larger molecular data sets ($>$200,000 characters and up to $\sim $100 taxa) showing node stability even with $\geqq100\times $ upweighting of morphological data. Accordingly, our analyses have three strong messages. 1) The assumption that genomic data will automatically "swamp out" morphological data is not always true for the SOM question. Morphological data have a strong influence in our analyses of combined data sets, even when outnumbered thousands of times by molecular data. Morphology therefore should not be counted out a priori. 2) We here quantify for the first time how the stability of the SOM node improves for several genomic data sets when the taxon sampling is increased. 3) The patterns of "flipping points" (i.e., the weighting of morphological data it takes to change the inferred SOM) carry information about the phylogenetic stability of matrices. The weighting space is an innovative way to assess comparability of data sets that could be developed into a new sensitivity analysis tool. [Metazoa; Morphology; Phylogenomics; Weighting.]., (© The Author(s) 2020. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.)

Placozoans, nonbilaterian animals with the simplest known metazoan bauplan, are currently classified into 20 haplotypes belonging to three genera, Polyplacotoma, Trichoplax, and Hoilungia. The latter two comprise two and five clades, respectively. In Trichoplax and Hoilungia, previous studies on six haplotypes belonging to four different clades have shown that their mtDNAs are circular chromosomes of 32-43 kb in size, which encode 12 protein-coding genes, 24 tRNAs, and two rRNAs. These mitochondrial genomes (mitogenomes) also show unique features rarely seen in other metazoans, including open reading frames (ORFs) of unknown function, and group I and II introns. Here, we report seven new mitogenomes, covering the five previously described haplotypes H2, H17, H19, H9, and H11, as well as two new haplotypes, H23 (clade III) and H24 (clade VII). The overall gene content is shared between all placozoan mitochondrial genomes, but genome sizes, gene orders, and several exon-intron boundaries vary among clades. Phylogenomic analyses strongly support a tree topology different from previous 16S rRNA analyses, with clade VI as the sister group to all other Hoilungia clades. We found small inverted repeats in all 13 mitochondrial genomes of the Trichoplax and Hoilungia genera and evaluated their distribution patterns among haplotypes. Because Polyplacotoma mediterranea (H0), the sister to the remaining haplotypes, has a small mitochondrial genome with few small inverted repeats and ORFs, we hypothesized that the proliferation of inverted repeats and ORFs substantially contributed to the observed increase in the size and GC content of the Trichoplax and Hoilungia mitochondrial genomes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure-function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines.

Dicer proteins are highly conserved, are present in organisms ranging from plants to metazoans, and are essential components of the RNA interference pathway. Although the complement of Dicer proteins has been investigated in many "higher" metazoans, there has been no corresponding characterization of Dicer proteins in any early-branching metazoan. We cloned partial cDNAs of genes belonging to the Dicer family from the anthozoan cnidarian Nematostella vectensis and two distantly related haplotypes (species lineages) of the Placozoa (Trichoplax adhaerens 16S haplotype 1 [H1] and Placozoa sp. [H2]). We also identified Dicer genes in the hydrozoan Hydra magnipapillata and the demosponge Amphimedon queenslandica with the use of publicly available sequence databases. Two Dicer genes are present in each cnidarian species, whereas five Dicer genes each are found in the Porifera and Placozoa. Phylogenetic analyses comparing these and other metazoan Dicers suggest an ancient duplication event of a "Proto-Dicer" gene. We show that the Placozoa is the only known metazoan phylum which contains both representatives of this duplication event and that the multiple Dicer genes of the "basal" metazoan phyla represent lineage-specific duplications. There is a striking diversity of Dicer genes in basal metazoans, in stark contrast to the single Dicer gene found in most higher metazoans. This new data has allowed us to formulate new hypotheses regarding the evolution of metazoan Dicer proteins and their possible functions in the early diverging metazoan phyla. We theorize that the multiple placozoan Dicer genes fulfill a specific biological requirement, such as an immune defense strategy against viruses.

A detailed annotation of non-protein coding RNAs is typically missing in initial releases of newly sequenced genomes. Here we report on a comprehensive ncRNA annotation of the genome of Trichoplax adhaerens, the presumably most basal metazoan whose genome has been published to-date. Since blast identified only a small fraction of the best-conserved ncRNAs--in particular rRNAs, tRNAs and some snRNAs--we developed a semi-global dynamic programming tool, GotohScan, to increase the sensitivity of the homology search. It successfully identified the full complement of major and minor spliceosomal snRNAs, the genes for RNase P and MRP RNAs, the SRP RNA, as well as several small nucleolar RNAs. We did not find any microRNA candidates homologous to known eumetazoan sequences. Interestingly, most ncRNAs, including the pol-III transcripts, appear as single-copy genes or with very small copy numbers in the Trichoplax genome.

Cnidarians display most of the characters considered as milestones of metazoan evolution. Whereas a tissue-level organization was probably already present in the multicellular common ancestor of all animals, the Urmetazoa, the emergence of important animal features such as bilateral symmetry, triploblasty, a polarized nervous system, sense organs (eyes, statocysts), and a (chitinous or calcium-based) continuous skeleton can be traced back before the divergence between cnidarians and bilaterians. Modularity and metamery might be also regarded as two faces of the same medal, likely involving conserved molecular mechanisms ruling animal body architectures through regional specification of iterated units. Available evidence indicates that the common ancestor of cnidarians and bilaterians, the UrEumetazoa, was a surprisingly complex animal with nerve cell differentiation. We suggest that paedomorphic events in descendants of this ancestor led to the array of diversity seen in the main extant animal phyla. The use of molecular analyses and identifying the genetic determinants of anatomical organizations can provide an integrative test of hypotheses of homologies and independent evidence of the evolutionary relationships among extant taxa.

Unraveling the root of the metazoan tree of life has been a difficult task since the time of Haeckel and the invention of phylogenetics. Even considerable amounts of recent molecular data have not provided a generally accepted answer. Here, we review the major problems of this phylogenetic conundrum and provide some directions for solving it.

The past two years have seen a dramatic increase in the available data on gene sequence and gene expression for cnidarians and other "lower" Metazoa, and a flurry of recent papers has drawn on these to address the origins of bilaterality. Cnidarian homologs of many genes that play key roles in the specification of both the A/P and D/V axes of bilaterians have been characterized, and their patterns of expression determined. Some of these expression patterns are consistent with the possibility of conservation of function between Cnidaria and Bilateria, but others clearly differ. Moreover, in some cases very different interpretations have been made on the basis of the same, or similar, data. In part, these differences reflect the inevitable uncertainties associated with the depth of the divergence between cnidarians and bilaterians. In this article, we briefly summarize the cnidarian data on gene expression and organization relevant to axis formation, the varying interpretations of these data, and where they conflict. Our conclusion is that the presently available data do not allow us to unequivocally homologize the single overt axis of cnidarians with either of the bilaterian axes.

In order to address this subject further and to assess progress in the examination of animal origins and transitions, an international group of scientists was convened at the Society for Comparative Biology meeting in January 2007.

A newly compiled data set of nearly complete sequences of the large subunit of the nuclear ribosome (LSU or 28S) sampled from 31 diverse medusozoans greatly clarifies the phylogenetic history of Cnidaria. These data have substantial power to discern among many of the competing hypotheses of relationship derived from prior work. Moreover, LSU data provide strong support at key nodes that were equivocal based on other molecular markers. Combining LSU sequences with those of the small subunit of the nuclear ribosome (SSU or 18S), we present a detailed working hypothesis of medusozoan relationships and discuss character evolution within this diverse clade. Stauromedusae, comprising the benthic, so-called stalked jellyfish, appears to be the sister group of all other medusozoans, implying that the free-swimming medusa stage, the motor nerve net, and statocysts of ecto-endodermal origin are features derived within Medusozoa. Cubozoans, which have had uncertain phylogenetic affinities since the elucidation of their life cycles, form a clade-named Acraspeda-with the scyphozoan groups Coronatae, Rhizostomeae, and Semaeostomeae. The polyps of both cubozoans and hydrozoans appear to be secondarily simplified. Hydrozoa is comprised by two well-supported clades, Trachylina and Hydroidolina. The position of Limnomedusae within Trachylina indicates that the ancestral hydrozoan had a biphasic life cycle and that the medusa was formed via an entocodon. Recently hypothesized homologies between the entocodon and bilaterian mesoderm are therefore suspect. Laingiomedusae, which has often been viewed as a close ally of the trachyline group Narcomedusae, is instead shown to be unambiguously a member of Hydroidolina. The important model organisms of the Hydra species complex are part of a clade, Aplanulata, with other hydrozoans possessing direct development not involving a ciliated planula stage. Finally, applying phylogenetic mixture models to our data proved to be of little additional value over a more traditional phylogenetic approach involving explicit hypothesis testing and bootstrap analyses under multiple optimality criteria. [18S; 28S; Cubozoa; Hydrozoa; medusa; molecular systematics; polyp; Scyphozoa; Staurozoa.].

In comparative studies using model organisms, extant taxa are often referred to as basal. The term suggests that such taxa are descendants of lineages that diverged early in the history of some larger taxon. By this usage, the basal metazoans comprise just four phyla (Placozoa, Porifera, Cnidaria, and Ctenophora) and the large clade Bilateria. We advise against this practice because basal refers to a region at the base or root of a phylogenetic tree. Thus, referring to an extant taxon or species as basal, or as more basal than another, can be misleading. While much progress has been made toward understanding some of the phylogenetic relationships within these groups, the relationships among them are still largely not known with certainty. Thus, sound inferences from comparative studies of model organisms demand continued illumination of phylogeny. Hypotheses about the mechanisms underlying metazoan evolution can be drawn from the study of model organisms in Cnidaria, Ctenophora, Placozoa, and Porifera, but it is clear that these model organisms are likely to be derived in many respects. Therefore, testing these hypotheses requires the study of yet additional model organisms. The most effective tests are those that investigate model organisms with phylogenetic positions among two sister groups comprising a larger clade of interest.

Pax genes play key regulatory roles in embryonic and sensory organ development in metazoans but their evolution and ancestral functions remain widely unresolved. We have isolated a Pax gene from Placozoa, beside Porifera the only metazoan phylum that completely lacks nerve and sensory cells or organs. These simplest known metazoans also lack any kind of symmetry, organs, extracellular matrix, basal lamina, muscle cells, and main body axis. The isolated Pax gene from Trichoplax adhaerens harbors a paired domain, an octapeptide, and a full-length homeodomain. It displays structural features not only of PaxB and Pax2/5/8-like genes but also of PaxC and Pax6 genes. Conserved splice sites between Placozoa, Cnidaria, and triploblasts, mark the ancient origin of intron structures. Phylogenetic analyses demonstrate that the Trichoplax PaxB gene, TriPaxB, is basal not only to all other known PaxB genes but also to PaxA and PaxC genes and their relatives in triploblasts (namely Pax2/5/8, Pax4/6, and Poxneuro). TriPaxB is expressed in distinct cell patches near the outer edge of the animal body, where undifferentiated and possibly multipotent cells are found. This expression pattern indicates a developmental role in cell-type specification and/or differentiation, probably in specifying-determining fiber cells, which are regarded as proto-neural/muscle cells in Trichoplax. While PaxB, Pax2/5/8, and Pax6 genes have been linked to nerve cell and sensory system/organ development in virtually all animals investigated so far, our study suggests that Pax genes predate the origin of nerve and sensory cells.

The phylum Placozoa is represented by a single known species, Trichoplax adhaerens, a tiny marine organism that represents the most simple metazoan bauplan. Because of the latter, placozoans were originally considered the most basal metazoan phylum. A misinterpretation of the life cycle at the turn of the century and some more recent molecular phylogenetic analyses have placed Trichoplax as a derived species within the Cnidaria. The latter hypothesis assumes that the primitive organization of the Placozoa is the result of secondary reduction. Here we compare the molecular morphology of the predicted 16S rDNA structure and the mitochondrial genome between Trichoplax and representatives of all four cnidarian classes. Trichoplax shares a circular mtDNA molecule as a plesiomorphy with all other metazoans except for the derived cnidarian classes Hydrozoa, Scyphozoa, and Cubozoa. The predicted secondary structure of the 16S rRNA molecule differs substantially between Trichoplax and cnidarians, particularly with respect to the number and length of stem and loop regions. The new molecular morphological characters provide compelling evidence that Trichoplax is not a derived (medusozoan) cnidarian. Furthermore, it was found that the mitochondrial genome in Cubozoa consists of four linear molecules instead of a single circular molecule or two linear molecules, suggesting that the cubozoans may represent the most derived cnidarian group.